The Eggers laboratory proposes to develop and test a thermodynamic framework for aqueous reaction equilibria that treats the solvent as a co-reactant. The new approach emphasizes the fact that the thermodynamic activity of water is a variable; the average free energy of water is a complicated function of the number and orientation of hydrogen bonds between neighboring molecules (enthalpy) and the randomness of their positions (entropy), and both of these thermodynamic properties are greatly influenced by the presence of a surface or solute. When the aqueous solute is a chemical reactant, it becomes appropriate to include water in the balanced reaction. In the case of binding reactions, a subset of hydration water is released to the bulk phase, and, consequently, there must be a term in the governing equation that accounts for the average free energy of bulk water. Because binding events are fundamental to many biological processes, this research is expected to enhance our understanding of the role of water in cell biology and pharmacology. Calorimetry and microscale thermophoresis will be employed to measure the desolvation energy of specific binding reactions, as guided by new thermodynamic equations derived by the PI. Selected binding reactions will be characterized in the presence of secondary solutes to determine if changes in binding affinity are fully explained by changes in the free energy of the bulk water. This research provides an alternative view for understanding the effects of solutes on biological equilibria, a view that may be deemed as more intuitive and more widely applicable than current models. The thermodynamic framework provides a fresh approach for characterizing concentrated, nonideal solutions, as relevant for understanding the driving forces behind molecular interactions in a cell or tissue. In addition, the experimental data obtained from this project may be exceedingly useful to computational scientists in developing realistic force fields for water at interfaces, an important consideration for computer simulations of biological processes.